The challenge in leukemia research is to develop effective therapies that decrease toxicity, maintain remission and prolong survival. Recently, the proteasome has become an attractive molecular target in cancer therapy. Earlier research shows that inhibition of the proteasome with proteasome inhibitors results in apoptosis in a variety of cancer cells. This project will test the hypothesis that differential inhibition of the proteasome p subunits, for example by a novel proteasome inhibitor, NPI-0052, or by down regulation of p subunit expression leads to activation of caspases, which ultimately results in apoptosis. The specific aims of this study are: 1Characterize the mechanism by which NPI-0052 induces apoptosis. NPI-0052 is a novel, clinically relevant, orally active, non-peptide small molecule proteasome inhibitor that inhibits the three proteolytic activities localized to the p5, p2 and pi subunits of the proteasome.Our preliminary data indicates that inhibiting the proteolytic activities with NPI-0052 induces caspase-8 activation, resulting in apoptosis in leukemia cells. Thus, we set out to assess the requirement of caspase-8 activation in NPI-0052-induced apoptotic events using a caspase-8 deficient cell line. Careful time and dose response experiments will be conducted to address the order of events, including caspase activation, for NPI-0052 to induce apoptosis. 2) Down regulate specific p proteasomal subunits to investigate their effects on the induction of the apoptotic process. Our initial findings with NPI-0052 suggested that additional studies are needed to dissect what aspect of proteasome inhibition, such as what p subunits are responsible and to what degree of inhibition of these subunits are needed, to induce activation of the apoptotic process. Thus, the second part of the proposed research examines the role of proteasomal p subunits in triggering apoptotic events such as activation of caspases. Silencing of specific proteasome p subunits will be achieved with RNA interference and different classic apoptotic end points will be examined. The requirement of caspase-8 in proteasomal p subunit down regulation-induced apoptosis will also be investigated using caspase peptide inhibitors. Improved understanding of the relationship between inhibition of specific proteasomal p subunits and triggering of the apoptotic cascade will provide insight into therapeutic strategies, such as NPI-0052, to target the proteasome in malignancies such as leukemia. The goal of this research is to reveal opportunities to improve and develop better therapeutic strategies in malignancies such as leukemia.